Shakeel M, Choi Y, Yoon M.Cryptorchidism affects spermatogenesis and testis development, often resulting in stallion subfertility/infertility. This study aims to identify the specific germ cells impacted by cryptorchism in stallions. In a previous study, we found that PGP9.5 and VASA are molecular markers expressed in different germ cells within stallions. Herein, we assessed the heat stress-induced response of spermatogonial stem cells (SSCs) in the seminiferous tubules (ST) of cryptorchid stallion testes (CST) and normal stallion testes (NST). This goal was accomplished by comparing PGP9.5 and VASA expression pattern...
Cinone M, Albrizio M, Guaricci AC, Lacitignola L, Desantis S.Heat shock proteins are the most evolutionarily conserved protein families induced by stressors including hyperthermia. In the context of pathologies of the male reproductive tract, cryptorchidism is the most common genital defect that compromises the reproductive potential of the male because it induces an increase in intratesticular temperature. In equine species, cryptorchidism affects almost 9 % of newborns and few studies have been carried out on the molecular aspects of the retained testis. In this study, the expression pattern of HSP60, 70, and 90 in abdominal and inguinal testes, in t...
da Silva CMB, Cano FEM, Gaitskell-Phillips G, Vega FJP.Flow cytometry is a powerful tool for the analysis of cell samples formed of multipopulations, such as spermatozoa. In recent years, multiparametric cytometers have evolved, allowing the study of different cellular characteristics, such as protein expression, DNA analysis, or mitochondrial activity. Whether using traditional fluorescent dyes or fluorophore-conjugated antibodies, each cell or cellular component is individually stained, the sample is analyzed at high velocities, and then is displayed and interpreted in a dot-plot. We hereby describe the procedure to perform a multiparametric flo...
Hamed MA, Amin YA, Mohamed RH, El-Adl M, Bazeed SM, Elnegiry AA, Shawki HH, Al-Lethie AA.Chemical castration of male animals is an alternative to surgical castration for inducing azoospermia, consequent sterility. Intra-testicular injection of zinc gluconate has been used for chemical castration in several animal species. However, its application to equine species, such as donkeys, has yet to be reported. This study aimed to evaluate the use of zinc gluconate for the chemical castration of male donkeys and to compare its effectiveness relative to routine surgical castration. For this purpose, investigations of serum testosterone and anti-Müllerian hormone levels, testicular ultra...
Ku00f6hne M, Kirch F, Tu00f6nissen A, Martinsson G, Rabe U, Sieme H, Schuberth HJ.To improve accuracy in evaluating stallion ejaculates, an antibody-based, flow cytometric assay for the detection and identification of leukocyte subpopulations (CD4-, CD8-, CD21-, CD172a-positive cells) in stallion semen (n = 12) was established. For establishment of the assay, native semen was supplemented with blood leukocytes (control: 20% leukocytes, 80% sperm cells) and analysed by flow cytometry. Adding antioxidants (ascorbic acid and butylated hydroxytoluol) to semen immediately after collection inhibited rapid death of lymphoid cells in sperm leukocyte mixtures. In control set-ups...
Shakeel M, Yoon M.This study investigated the effect of heat stress on stallion testicular cells (TCs) and the effect of insulin-like growth factor (IGF)-1 on TC viability, proliferation, and apoptosis, including different stages of germ cells. TCs were divided into control or treatment groups with 0.01, 0.1, 1, 10, and 100 ng/mL of recombinant human IGF-1 (rhIGF-1) for 24 h at 34 °C and 37 °C. The population and viability were measured before and after treatment. The effects of rhIGF-1 on TC viability, proliferation, and apoptosis were determined using RT-qPCR. Proliferating cell nuclear antigen (PCNA) a...
Laseca N, Cu00e1novas u00c1, Valera M, Id-Lahoucine S, Perdomo-Gonzu00e1lez DI, Fonseca PAS, Demyda-Peyru00e1s S, Molina A.The phenomenon in which the expected Mendelian inheritance is altered is known as transmission ratio distortion (TRD). The TRD analysis relies on the study of the transmission of one of the two alleles from a heterozygous parent to the offspring. These distortions are due to biological mechanisms affecting gametogenesis, embryo development and/or postnatal viability, among others. In this study, TRD phenomenon was characterized in horses using SNP-by-SNP model by TRDscan v.2.0 software. A total of 1,041 Pura Raza Español breed horses were genotyped with 554,634 SNPs. Among them, 277 horses ge...
Cavalcanti TP, Pereira AG, Bezerra LGP, Moreira SSJ, da Silva AM, Matos YG, Jorge-Neto PN, Silva AR.The use of assisted reproductive techniques, such as chilled semen, contributes to the maintenance and genetic improvement of canine breeding. The INRA-96 extender is a commercially available, chemically defined medium that was initially developed for the preservation of equine semen and exhibits preservation potential in the canine species. This research aims to evaluate the INRA-96 extender as an alternative for the short-term preservation of canine semen in terms of sperm quality parameters such as motility and kinetic parameters, integrity and functionality of the plasma membrane in fresh ...
Shakeel M, Yoon M.The threat posed by increased surface temperatures worldwide has attracted the attention of researchers to the reaction of animals to heat stress. Spermatogenesis in animals such as stallions is a temperature-dependent process, ideally occurring at temperatures slightly below the core body temperature. Thus, proper thermoregulation is essential, especially because stallion spermatogenesis and the resulting spermatozoa are negatively affected by increased testicular temperature. Consequently, the failure of thermoregulation resulting in heat stress may diminish sperm quality and increase the li...
Muu00f1oz E, Castro M, Aguila L, Contreras MJ, Fuentes F, Arias ME, Felmer R.Sperm sexing is a technology that can generate great economic benefits in the animal production sector. Techniques such as sex-sorting promise over 90% accuracy in sperm sexing. However, for the correct standardization of the technique, some laboratory methodologies are required. The present manuscript describes in detail a standardized equine sperm sex-sorting protocol using an absolute qPCR-based methodology. Furthermore, the results of absolute qPCR were implemented and validated by generating equine/bovine heterologous embryos by intracytoplasmic sperm injection (ICSI) of presumably sexed ...
Mogielnicka-Brzozowska M, Wou017aniak MJ, Cichowska AW, Fraser L, Kraziu0144ski B, Strzezek R, Zieliu0144ska D.Seminal plasma (SP) proteins are responsible for sperm functional quality. Developing a reliable method to determine the degree of oxidative damage of these proteins is important for establishing semen fertilizing ability. The main aim of the study was to verify the applicability of protein carbonyl derivatives measurement in the SP of canine and stallion, using a method with 2,4-dinitrophenylhydrazine (DNPH). The research material consisted of ejaculates obtained from eight English Springer Spaniels, and from seven half-blood stallions during the breeding and non-breeding season. The content ...
Gonu00e7alves AR, Telo da Gama L, Antunes L, Guimaru00e3es H, Bliebernicht M, Duarte JC, Cosinha C, Duarte Rego B, Ferro da Costa P, Guimaru00e3es T....The objectives of this study were to establish baseline information for seminal traits in Lusitano stallions, to assess the impact of inbreeding, interval between collections and age on semen quality during the breeding and non-breeding seasons, and to estimate the corresponding genetic parameters. A total of 2129 ejaculates by 146 Lusitano stallions used for artificial insemination, obtained from four equine reproduction centers distributed throughout Portugal, over a period of 14 years (2008-2021), were included in the study. The seminal traits analyzed, and the corresponding means and stand...
Miller LMJ, Woodward EM, Campos JR, Squires EL, Troedsson MHT.Sperm protein at 22 kDa has been associated with fertility. Objective: The objectives of this study were to determine (1) the localization pattern of SP22 on ejaculated and caudal epididymal equine spermatozoa and in epididymal fluid, and to (2) characterize SP22 protein and mRNA expression in testicular and epididymal tissues in response to heat-induced testicular degeneration. Methods: Semen was collected before and after hemi-castration, as well as prior to and following insulation of the remaining testes, and tissue specimens were collected for analysis. Results: Histopathology confirmed ...
de Moura TCM, Arruda LCP, Cahu00fa TB, Bezerra RS, Carneiro GF, Guerra MMP.Semen cryopreservation ensures the storage of stallion genetics for an unlimited time. The improvement of extenders with new antioxidant substances can optimize the properties of post-thawed semen. The study aimed to investigate the addition effect of medium-molecular-weight carboxymethylchitosan (CQm) derivates to freezing diluent of stallion sperm after freezinf/thawing. Twice a week, five ejaculates of four stallions were obtained, totalizing 20 ejaculates. Semen was diluted in commercial freezing extender (Botucrio) supplemented with CQm: control (0), 0.75, 1.5, and 3 mg/mL. Samples were ...
Alim S, Laitaoja M, Pawar SS, Talluri TR, Ju00e4nis J, Swamy MJ.Major proteins of the seminal plasma in a variety of mammals such as bovine PDC-109, equine HSP-1/2, and donkey DSP-1 contain fibronectin type-II (FnII) domains and are referred to as FnII family proteins. To further our understanding on these proteins, we carried out detailed studies on DSP-3, another FnII protein of donkey seminal plasma. High-resolution mass-spectrometric studies revealed that DSP-3 contains 106 amino acid residues and is heterogeneously glycosylated with multiple acetylations on the glycans. Interestingly, high homology was observed between DSP-1 and HSP-1 (118 identical r...
Pozor MA, Benson SM, Macpherson ML, Kelleman AA.Transplantation of stem cells into dysfunctional testes is currently being investigated as a therapeutic option for men and stallions with advanced testicular degeneration. This series of "proof of concept" studies aimed to identify a safe and efficient method of inducing severe testicular degeneration to create an optimal equine recipient model for intratesticular stem cell transplantation (SCT). Two ex vivo and two in vivo experiments were conducted. At first, forty testes obtained from castrations were used to identify an effective therapeutic ultrasound (TUS) device and the protocol for ...
Hensel B, Jakop U, Schmicke M, Schru00f6ter F, Jung M, Schulze M.Artificial insemination (AI) is commonly used in the equine industry to enhance the genetic value in breeding programs and to effectively utilize ejaculates. Many stallions are used as breeding stallions as well as in high-level sports competitions to improve their market value. The goal of the present study was to investigate whether this dual use of stallions influences the animals´ stress levels and/or the quality of their ejaculates. For this purpose, 18 stallions were grouped into two categories: breeding stallions with (BSC = breeding stallion competition), and breeding stallions with...
Yu J, Wang Z, Wang F, Yang G, Cheng J, Ji C, Li M, Liu B, Wang Y, Dang R.Testicular development and spermatogenesis are tightly regulated by the number of genes and noncoding genes, and mRNAs and lncRNAs play vital roles in regulating posttranscriptional gene expression. However, mRNAs and lncRNAs have not been systematically identified in the testes of donkeys. In this study, mRNA and lncRNA expression profiles in the testes of DeZhou donkeys between 2 months and 2 years of age were comprehensively analysed by RNA sequencing. We identified 56,605 lncRNAs and 61,857 mRNAs by gene expression analysis, and 21,845 lncRNAs (p < .05) and 14,109 mRNAs (p < .0...
Freitas ML, Viana JHM, Dode MAN, Maggiotto SR, Pivato I, Braga TRC, Lim AIPG, de Oliveira RA.The effects of seasonality on the reproduction of stallions vary based on the latitude. Although previous studies have shown the influence of seasonality in raw semen quality in south-eastern Brazil, data regarding the influence of seasonality in cooled and frozen stored semen in Brazil is limited. Therefore, in this study, we have analysed if seasonality influences the hormone production (i.e., cortisol and testosterone), spermatogenesis, and quality of fresh, cooled, and frozen semen of stallions in central Brazil, and established the season most suitable for semen cryopreservation in a lati...
Al-Kass Z, Ntallaris T, Morrell JM, Johannisson A.Although previous studies have examined the relationship between the sperm DNA fragmentation index and fertility in stallions, other aspects of chromatin structure or packaging and fertility have not been explored. In the present study, relationships between fertility and DNA fragmentation index, protamine deficiency, total thiols, free thiols and disulfide bonds in stallion spermatozoa were investigated. Ejaculates (n = 36) were collected from 12 stallions and extended to prepare semen doses for insemination. One dose from each ejaculate was sent to the Swedish University of Agricultural Sc...
Assumpu00e7u00e3o TI, Lanu00e7oni R, Foschini M, Vieira CS.This study aimed to select high-quality spermatozoa by sperm separation by magnetic activation of the fresh equine semen, compared to density gradient centrifugation and evaluating cell quality after selection. The semen of 10 stallions was collected by the artificial vagina technique. The samples analyzed were: (1) fresh semen; (2) density gradient centrifugation (DGC); (3) separation by magnetic activation (MASS) (nonapoptotic portion NAP); (4) separation by MASS (apoptotic portion-APT). Was analyzed: motility (light microscopy), concentration (Neubauer chamber), semen morphology (humid cham...
Aitken RJ, Lambourne S, Medica AJ.A capacity to predict the likelihood of pregnancy following natural matings would be of considerable benefit to the Thoroughbred horse breeding industry. In this article, we describe a strategy for achieving this outcome through the analysis of dismount samples, that achieved an overall accuracy of 94.6%. The purpose of this study was to determine whether the analysis of dismount semen samples from Thoroughbred stallions could be used to predict whether a given mating would result in a pregnancy. The analysis was based on 143 matings of 141 mares by a cohort of 7 Thoroughbred stallions over a ...
Aparicio IM, Rojo-Domu00ednguez P, Castillejo-Rufo A, Peu00f1a FJ, Tapia JA.Despite its importance in somatic cells and during spermatogenesis, little is known about the role that autophagy may play in ejaculated spermatozoa. Our aim was to investigate whether the molecular components of autophagy, such as microtubule-associated protein 1 light chain 3 (LC3), are activated in stallion spermatozoa during the capacitation and acrosome reaction and if this activation could modulate these biological processes. To analyze the autophagy turnover, LC3I and LC3II proteins were assessed by western blotting, and the ratio between both proteins (LC3II/LC3I) was calculated. In so...
Prasinou P, De Amicis I, Fusaro I, Bucci R, Cavallini D, Parrillo S, Caputo M, Gramenzi A, Carluccio A.Fatty acid-based lipidomic analysis has been widely used to evaluate health status in human medicine as well as in the veterinary field. In equine species, there has been a developing interest in fertility and sperm quality. Fatty acids, being the principal components of the membranes, play an active role in the regulation of the metabolic activities, and their role on spermiogenesis seems to be of great importance for the resulting quality of the sperm and, thus, fertility. With the application of widely used lipidomic techniques, the aim of this study was to evaluate: (a) the fatty acid cont...
Peu00f1a FJ, Gibb Z.The growing understanding of the mechanisms regulating redox homeostasis in the stallion spermatozoa, together with its interactions with energetic metabolism, is providing new clues applicable to the improvement of sperm conservation in horses. Based on this knowledge, new extenders, adapted to the biology of the stallion spermatozoa, are expected to be developed in the near future. The preservation of semen either by refrigeration or cryopreservation is a principal component of most animal breeding industries. Although this procedure has been successful in many species, in others, substantia...
Samir H, ElSayed MI, Radwan F, Hedia M, Hendawy H, Hendawy AO, Elbadawy M, Watanabe G.In all organs, control of blood flow is important but might be particularly critical for testicular functions. This is because of the very low oxygen concentration and high metabolic rate of the seminiferous tubules, the physiological temperature of the testis, and its location outside the abdominal cavity. Many factors affect the characteristics of TBF in farm and companion animals, such as environment (thermal and seasonal effects) and physiological (species, breeds, age, body weight, and sexual maturity). Thermal environment stress has detrimental effects on spermatogenesis and consequently...
Hernu00e1ndez-Avilu00e9s C, Ramu00edrez-Agu00e1mez L, Varner DD, Love CC.During the fertilization process, the interaction between the sperm and the oocyte is mediated by a process known as acrosomal exocytosis (AE). Although the role of the sperm acrosome on fertilization has been studied extensively over the last 70 years, little is known about the molecular mechanisms that govern acrosomal function, particularly in species other than mice or humans. Even though subfertility due to acrosomal dysfunction is less common in large animals than in humans, the evaluation of sperm acrosomal function should be considered not only as a complementary but a routine test whe...
Waheed MM, Meligy A, Alhaider AK, Ghoneim IM.Seminal plasma contains several microelements like Zn, Fe, Se, and Cu that affect sperm motility and male fertility. Biochemical evaluation of seminal plasma trace elements is important for assessing fertility and diagnosing male infertility. Unassigned: The present study was designed to evaluate the effect of seminal fluid trace elements on sperm parameters and fertility in Arabian horses. Unassigned: Ninety-four ejaculates from 25 Arabian stallions (4-27 years old) were used to investigate the effect of seminal fluid trace elements on semen parameters and fertility. Data divided according to...
Bazzano M, Zhu C, Laus F, Giambattista AD, Laghi L.The application of the 'omics' studies in the field of animal reproduction has been aimed at identifying novel biomarkers of fertility since the last few years. When assessing reproductive efficiency in horses, breed should also be taken into account as it can influence semen quality and fertility. Considering the growing interest in metabolomic analysis to evaluate male fertility, we aimed to investigate the metabolomic profile of seminal plasma in two different horse breeds. Twelve healthy stallions, n.6 American Quarter Horse (AQH) and n.6 Italian Draft Horse (IDH) stallions, regularly used...
Gautier C, Aurich J, Kaps M, Okada CTC, Wagner LH, Melchert M, Aurich C.In stallions temporarily not intended for breeding, reversible suppression of testicular function by vaccination against GnRH can be of interest. In the present study, effects of GnRH agonist treatment on the resumption of testicular function after GnRH vaccination were investigated. Testis size, testosterone release, semen characteristics and behavior were evaluated. We hypothesized that GnRH agonist treatment would restore testicular function. Shetland stallions were assigned to an experimental and a control group (n = 6 each). Experimental stallions were GnRH-immunized twice, four weeks a...
Johnson L, Thompson DL, Varner DD.Testicular function is under the control of expression and repression of several genes and gene products, and many of these works through Sertoli cells. The capability of Sertoli cells to regulate spermatogenesis is dependent on Sertoli cell functions and Sertoli cell number. Sertoli cell number has long been thought to be stable in adults with no proliferation of Sertoli cells once adult numbers have been reached. However, adult horses do not have stable Sertoli cell numbers, and new studies indicate that adult Sertoli cells can be made to re-enter mitotic phase under certain experimental con...
Su TW, Choi I, Feng J, Huang K, McLeod E, Ozcan A.We report the discovery of an entirely new three-dimensional (3D) swimming pattern observed in human and horse sperms. This motion is in the form of 'chiral ribbons', where the planar swing of the sperm head occurs on an osculating plane creating in some cases a helical ribbon and in some others a twisted ribbon. The latter, i.e., the twisted ribbon trajectory, also defines a minimal surface, exhibiting zero mean curvature for all the points on its surface. These chiral ribbon swimming patterns cannot be represented or understood by already known patterns of sperms or other micro-swimmers. The...
Druart X, Rickard JP, Mactier S, Kohnke PL, Kershaw-Young CM, Bathgate R, Gibb Z, Crossett B, Tsikis G, Labas V, Harichaux G, Grupen CG, de Graaf SP.Seminal plasma contains a large protein component which has been implicated in the function, transit and survival of spermatozoa within the female reproductive tract. However, the identity of the majority of these proteins remains unknown and a direct comparison between the major domestic mammalian species has yet to be made. As such, the present study characterized and compared the seminal plasma proteomes of cattle, horse, sheep, pig, goat, camel and alpaca. GeLC-MS/MS and shotgun proteomic analysis by 2D-LC-MS/MS identified a total of 302 proteins in the seminal plasma of the chosen mammali...
Ball BA.Oxidative stress is an important component of the cytopathology of equine spermatozoa undergoing storage as liquid or frozen semen. Damage to chromatin, membranes and proteins of sperm are important components of oxidative damage to sperm. Similarly, sperm are exposed to a variety of osmotic stresses during storage that result from exposure to hypertonic media or result as a consequence of osmotic changes induced during freezing. A number of changes induced during processing and storage of equine sperm also appear to induce apoptotic-like changes which may adversely affect sperm survival and f...
Rathi R, Honaramooz A, Zeng W, Turner R, Dobrinski I.Grafting of testis tissue from immature animals to immunodeficient mice results in complete spermatogenesis, albeit with varying efficiency in different species. The objectives of this study were to investigate if grafting of horse testis tissue would result in spermatogenesis, and to assess the effect of exogenous gonadotropins on xenograft development. Small fragments of testis tissue from 7 colts (2 week to 4 years of age) were grafted under the back skin of castrated male immunodeficient mice. For 2 donor animals, half of the mice were treated with gonadotropins. Xenografts were analyzed a...
Westhoff D, Kamp G.Evidence is provided that the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase is covalently linked to the fibrous sheath. The fibrous sheath is a typical structure of mammalian spermatozoa surrounding the axoneme in the principal piece of the flagellum. More than 90% of boar sperm glyceraldehyde 3-phosphate dehydrogenase activity is sedimented after cell disintegration by centrifugation. Detergents, different salt concentrations or short term incubation with chymotrypsin do not solubilize the enzyme, whereas digestion with trypsin or elastase does. Short term incubation with trypsin...
Gibb Z, Lambourne SR, Aitken RJ.The relationship between stallion fertility and oxidative stress remains poorly understood. The purpose of this study was to identify criteria for thoroughbred fertility assessment by performing a logistical regression analysis using "dismount" sperm parameters as predictors and weekly per-cycle conception rate as the dependent variable. Paradoxically, positive relationships between fertility and oxidative stress were revealed, such that samples that produced pregnancies exhibited higher rates of 8-hydroxy-2'-deoxyguanosine release (1490.2% vs. 705.5 pg/ml/24 h) and lower vitality (60.5% vs. 6...
Janeu010dka JE, Davis BW, Ghosh S, Paria N, Das PJ, Orlando L, Schubert M, Nielsen MK, Stout TAE, Brashear W, Li G, Johnson CD, Metz RP, Zadjali AMA....Dynamic evolutionary processes and complex structure make the Y chromosome among the most diverse and least understood regions in mammalian genomes. Here, we present an annotated assembly of the male specific region of the horse Y chromosome (eMSY), representing the first comprehensive Y assembly in odd-toed ungulates. The eMSY comprises single-copy, equine specific multi-copy, PAR transposed, and novel ampliconic sequence classes. The eMSY gene density approaches that of autosomes with the highest number of retained X-Y gametologs recorded in eutherians, in addition to novel Y-born and transp...
Plaza Davila M, Martin Muu00f1oz P, Tapia JA, Ortega Ferrusola C, Balao da Silva C C, Peu00f1a FJ.Mitochondria have been proposed as the major source of reactive oxygen species in somatic cells and human spermatozoa. However, no data regarding the role of mitochondrial ROS production in stallion spermatozoa are available. To shed light on the role of the mitochondrial electron transport chain in the origin of oxidative stress in stallion spermatozoa, specific inhibitors of complex I (rotenone) and III (antimycin-A) were used. Ejaculates from seven Andalusian stallions were collected and incubated in BWW media at 37 °C in the presence of rotenone, antimycin-A or control vehicle. Incubation...
Paria N, Raudsepp T, Pearks Wilkerson AJ, O'Brien PC, Ferguson-Smith MA, Love CC, Arnold C, Rakestraw P, Murphy WJ, Chowdhary BP.Studies of the Y chromosome in primates, rodents and carnivores provide compelling evidence that the male specific region of Y (MSY) contains functional genes, many of which have specialized roles in spermatogenesis and male-fertility. Little similarity, however, has been found between the gene content and sequence of MSY in different species. This hinders the discovery of species-specific male fertility genes and limits our understanding about MSY evolution in mammals. Here, a detailed MSY gene catalogue was developed for the horse--an odd-toed ungulate. Using direct cDNA selection from horse...
Das PJ, McCarthy F, Vishnoi M, Paria N, Gresham C, Li G, Kachroo P, Sudderth AK, Teague S, Love CC, Varner DD, Chowdhary BP, Raudsepp T.Mature mammalian sperm contain a complex population of RNAs some of which might regulate spermatogenesis while others probably play a role in fertilization and early development. Due to this limited knowledge, the biological functions of sperm RNAs remain enigmatic. Here we report the first characterization of the global transcriptome of the sperm of fertile stallions. The findings improved understanding of the biological significance of sperm RNAs which in turn will allow the discovery of sperm-based biomarkers for stallion fertility. The stallion sperm transcriptome was interrogated by analy...
Sailer BL, Jost LK, Evenson DP.Sperm from four mammalian species were analyzed by the sperm chromatin structure assay (SCSA) and the terminal deoxynucleotidyl transferase assay (TdTA) using flow cytometry. The SCSA quantitates the susceptibility of sperm nuclear DNA to in situ acid denaturation, while the TdTA quantitates the presence of endogenous DNA strand breaks in sperm nuclear chromatin. Correlations were seen between the percentage of sperm cells showing susceptibility to in situ acid denaturation and the percentage of cells showing the presence of DNA strand breaks for humans (r = 0.56, P = 0.004), rams (r = 0.84, P...
Chemineau P, Guillaume D, Migaud M, Thiu00e9ry JC, Pellicer-Rubio MT, Malpaux B.Farm mammals generally express seasonal variations in their production traits, thus inducing changing availability of fresh derived animal products (meat, milk and cheese) or performances (horses). This is due to a more or less marked seasonal birth distribution in sheep and goats, in horses but not cattle. Birth peak occurs at the end of winter-early spring, the most favourable period for the progeny to survive. Most species show seasonal variations in their ovulation frequency (presence or absence of ovulation), spermatogenic activity (from moderate decrease to complete absence of sperm prod...
Galli C, Duchi R, Colleoni S, Lagutina I, Lazzari G.Assisted reproductive techniques developed for cattle in the last 25 years, like ovum pick up (OPU), intracytoplasmic sperm injection (ICSI), and somatic cell nuclear transfer, have been transferred and adapted to buffalo and horses. The successful clinical applications of these techniques require both the clinical skills specific to each animal species and an experienced laboratory team to support the in vitro phase of the work. In cattle, OPU can be considered a consolidated technology that is rapidly outpacing conventional superovulation for embryo transfer. In buffalo, OPU represents the ...
Davila MP, Muu00f1oz PM, Bolau00f1os JM, Stout TA, Gadella BM, Tapia JA, da Silva CB, Ferrusola CO, Peu00f1a FJ.To investigate the hypothesis that oxidative phosphorylation is a major source of ATP to fuel stallion sperm motility, oxidative phosphorylation was suppressed using the mitochondrial uncouplers CCCP and 2,4,-dinitrophenol (DNP) and by inhibiting mitochondrial respiration at complex IV using sodium cyanide or at the level of ATP synthase using oligomycin-A. As mitochondrial dysfunction may also lead to oxidative stress, production of reactive oxygen species was monitored simultaneously. All inhibitors reduced ATP content, but oligomycin-A did so most profoundly. Oligomycin-A and CCCP also sign...
Sabeur K, Ball BA.Reactive oxygen species (ROS) play an important role in normal sperm function, and spermatozoa possess specific mechanisms for ROS generation via an NAD(P)H-dependent oxidase. The aim of this study was to identify the presence of an NADPH oxidase 5 (NOX5) in equine testis and spermatozoa. The mRNA of NOX5 was expressed in equine testis as detected by northern blot probed with human NOX5 cDNA and by RT-PCR. Immunoblotting with affinity purified alpha-NOX5 revealed one major protein in equine testis and other tissues. Immunolocalization of NOX5 showed labeling over the rostral sperm head with so...
Pesch S, Bergmann M, Bostedt H.Seminal plasma is very important for sperm metabolism as well as sperm function and survival and transport in the female genital tract. Analysis of enzyme activities and concentrations of elements can estimate integrity and function of sperm cell membranes. In man much data are available about biochemical analyses of seminal plasma. However, not many studies have been conducted in horses yet. We collected ejaculates from 72 stallions, measured the volume, obtained seminal plasma by centrifugation and examined spermatozoa with light microscopy for motility, concentration, for dead sperm and mor...
Colenbrander B, Gadella BM, Stout TA.Pregnancy rates in managed horse populations depend on the innate fertility of the mares and stallions involved and on the quality of breeding management. Of course, because a single stallion usually mates many mares, stallion fertility is a critical factor in the overall success of a breeding program. Unfortunately, accurate evaluation of stallion fertility per se requires a large number of normal mares to be mated and is necessarily retrospective. Rather, the ideal is to predict fertility in advance of the stallion's breeding career, and this is currently attempted by way of a thorough physi...
Arregui L, Rathi R, Zeng W, Honaramooz A, Gomendio M, Roldan ER, Dobrinski I.Xenografting of testis tissue from immature males from several mammalian species to immunodeficient mouse hosts results in production of fertilization-competent sperm. However, the efficiency of testis tissue xenografting from adult donors has not been critically evaluated. Testis tissue xenografting from sexually mature animals could provide an option to preserve the genetic material from valuable males when semen for cryopreservation cannot be collected. To assess the potential use of this technique for adult individuals, testes from adult animals of six species (pig, goat, cattle, donkey, h...
Russell LD, Ren HP, Sinha Hikim I, Schulze W, Sinha Hikim AP.Morphometric studies were performed on 12 mammalian species (degu, dog, guinea pig, hamster, human, monkey, mouse, opossum, rabbit, rat, stallion, and woodchuck) to determine volume density percentage (Vv%), volume (V), and numerical density (Nv) of seminiferous tubule components, especially those related to the Sertoli cell, and to make species comparisons. For most species, measurements were taken both from stages where elongate spermatids were deeply embedded within the Sertoli cell and from stages near sperm release where elongate spermatids were in shallow crypts within the Sertoli cell. ...
Canisso IF, Panzani D, Miru00f3 J, Ellerbrock RE.Donkeys are nonseasonal, polyestrous, territorial, and nonharem breeders. Although there are many similarities between horses and donkeys, there are also reproductive features that differ, from the longer cervix in the jenny to spermatogenic efficiency in the jack. Mules display reproductive cyclic activity but are rarely fertile. Frozen donkey semen has high pregnancy rates in mares, but lower rates in jennies. This article reviews key aspects of donkey and mule reproductive physiology, reproductive medicine, and assisted reproductive techniques that are useful for practitioners offering assi...
Steiner CC, Ryder OA.Prdm9 (Meisetz) is the first speciation gene discovered in vertebrates conferring reproductive isolation. This locus encodes a meiosis-specific histone H3 methyltransferase that specifies meiotic recombination hotspots during gametogenesis. Allelic differences in Prdm9, characterized for a variable number of zinc finger (ZF) domains, have been associated with hybrid sterility in male house mice via spermatogenic failure at the pachytene stage. The mule, a classic example of hybrid sterility in mammals also exhibits a similar spermatogenesis breakdown, making Prdm9 an interesting candidate to e...
Loomis PR, Graham JK.One of the challenges for those attempting to cryopreserve stallion spermatozoa is dealing with the stallion to stallion variability in the cryosurvival of their semen. In the dairy industry, each bull stud, essentially utilizes a single cryopreservation technique, and bulls that produce sperm that do not cryopreserve well using that technique are replaced by other bulls. However, replacing stallions is unlikely to prove acceptable to the equine industry, where specific genotypes are desired. Instead, to increase the number of stallions that can be effectively utilized for cryopreserved semen ...
Bedford-Guaus SJ, McPartlin LA, Xie J, Westmiller SL, Buffone MG, Roberson MS.Oocyte activation at fertilization is brought about by the testis-specific phospholipase C zeta (PLCZ), owing to its ability to induce oscillations in intracellular Ca(2+) concentration ([Ca(2+)](i)). Whereas this is a highly conserved mechanism among mammals, important species-specific differences in PLCZ sequence, activity, and expression have been reported. Thus, the objectives of this research were to clone and characterize the intracellular Ca(2+)-releasing activity and expression of equine PLCZ in sperm and testis. Molecular cloning of equine PLCZ yielded a 1914-bp sequence that translat...
Gallardo Bolau00f1os JM, Miru00f3 Moru00e1n u00c1, Balao da Silva CM, Morillo Rodru00edguez A, Plaza Du00e1vila M, Aparicio IM, Tapia JA....Apoptosis has been recognized as a cause of sperm death during cryopreservation and a cause of infertility in humans, however there is no data on its role in sperm death during conservation in refrigeration; autophagy has not been described to date in mature sperm. We investigated the role of apoptosis and autophagy during cooled storage of stallion spermatozoa. Samples from seven stallions were split; half of the ejaculate was processed by single layer centrifugation, while the other half was extended unprocessed, and stored at 5°C for five days. During the time of storage, sperm motility (C...
Ortega Ferrusola C, Gonzu00e1lez Fernu00e1ndez L, Salazar Sandoval C, Macu00edas Garcu00eda B, Rodru00edguez Martu00ednez H, Tapia JA, Peu00f1a FJ.In order to evaluate to what extent the changes that occur during cryopreservation involve the mitochondrial permeability transition pore (PT-pore), a specific inhibitor was used during the cryopreservation process of stallion spermatozoa. Four ejaculates from each of 7 stallions were frozen using a standard protocol. Before freezing, each ejaculate was split into three subsamples. The first was supplemented with 2.5 microM bongkrekic acid (BA) and the second with 5 microM BA. The third subsample served as control. The BA significantly reduced the percentage of spermatozoa depicting active cas...
Timoney PJ, McCollum WH, Murphy TW, Roberts AW, Willard JG, Carswell GD.The carrier state has been confirmed virologically in Thoroughbred and non-Thoroughbred stallions naturally infected with equine arteritis virus (EAV). Short-term or convalescent and long-term carriers occur. The frequency rate of the long-term carrier state in Thoroughbreds was high, averaging 33.9% among the three groups of stallions under study. While the convalescent carrier state only lasted a few weeks after clinical recovery, the long-term carrier state could persist for years. There was evidence, however, that not all such carriers might remain persistently infected for life. Carrier s...
Roser JF.It is well established in many mammalian species, including the horse that normal testicular function is dependent upon a functional hypothalamic-pituitary-testicular (HPT) axis, which involves classic feedback mechanisms. The major HPT hormones involved in the stallion are gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone (T), estrogens (Es) and inhibin (INH). Although prolactin (PRL) fluctuates with season in the stallion and both PRL and thyroid hormone (TH) affect reproduction in other male species, their effects on stallion r...
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